CLASSIFICATION AND IDENTIFICATION OF BACTERIA

School of Environmental Studies

Key Terms
Isolation (culture) Agar plate plate/colonies Liquid media After culture Biochemical (physiological) tests Genetic tests Sequencing, Polymerase chain reaction (PCR) DNA-DNA homology Chemical (e.g. fatty acid profiling) Immunological Direct detection (i.e. without culture) PCR Antigen detection Staining (e.g. Gram stain) Serology (antibody detection)

Identification & taxonomy Family Genus Species Type Strain

Classification

Strain: one single isolate or line Type: sub-set of species Species: related strains Genus: related species Family: related genera

Laboratory procedures employed in the identification of bacteria

1.Isolation of organism in pure culture 2.Bacterial colony morphology 3.Microscopic morphology and Staining reaction 4.Biochemical test 5.Serological procedure 6.Antibiotic sensitivity

Isolation of organism in Pure Culture

Pure culture (axenic culture) Population of cells arising from a single cell - the approach used for the isolation of organism depends upon the source of clinical specimen

Blood, spinal fluid and closed abscesses may yield almost pure bacterial culture
specimen of sputum, stool, materials from the skin and body orifices, waste water etc., usually contains mixture of organism - Spread plate, streak plate, and pour plate are

techniques used to isolate pure culture

Laboratory Cultivation

Cultivation is the process of growing microorganisms by taking bacteria from the infection site by some means of specimen collection and growing them in the artificial environment of the laboratory For the in vitro environment of the bacteria, required nutrients are supplied in a culture medium culture - organisms that grow and multiply in or on a culture media

Culture Medium - A liquid or gel designed to support the growth of microorganisms - 2 major types of growth media: - those used for cell culture, which use specific cell types derived from plants or animals - microbiological culture, which are used for growing microorganisms such as bacteria or yeast -The most common growth media for microorganisms are nutrient broths and agar plates -specialized media are sometimes required for microorganism and cell culture growth

Based on Chemical Composition Complex Media - Contain some ingredients of unknown composition and/or conc. - is a medium that contains: carbon source such as glucose for bacterial growth water various salts needed for bacterial growth a source of amino acids and nitrogen (e.g., beef, yeast extract) - Nutrient media contain all the elements that most bacteria need for growth and are non-selective, so they are used for the general cultivation and maintenance of bacteria kept in laboratory culture collections Defined or Synthetic Media -All components and their concentrations are known

Functional Types of Media Supportive or general purpose media

- E.g., Tryptic soy agar

- Support the growth of many microorganisms

Enriched media - General purpose media supplemented by blood or other special nutrients

Blood agar is an enriched medium in which nutritionally rich whole blood supplements the basic nutrients Chocolate agar is enriched with heat-treated blood (40-45C), which turns brown and gives the medium the color for which it is named

Selective media

- Favor the growth of only selected microorganisms and inhibit growth of others

eosin-methylene blue agar (EMB) that contains methylene blue toxic to Gram (+) bacteria, allowing only the growth of Gram (-) bacteria blood agar (used in strep tests), which contains beef heart blood that becomes transparent in the presence of hemolytic Streptococcus MacConkey agar for Gram-negative bacteria Mannitol Salt Agar (MSA) which is selective for Gram (+) bacteria and differential for mannitol

Differential media Distinguish between different groups of microorganisms based on their biochemical characteristics growing in the presence of specific nutrients or indicators (such as neutral red, phenol red, eosin y, or methylene blue) added to the medium to visibly indicate the defining characteristics of a microorganism Ex. Blood agar differentiates hemolytic versus non-hemolytic bacteria
MacConkey agar - lactose fermenters versus non-fermenters Eosin methylene blue (EMB), which is differential for lactose and sucrose fermentation Mannitol Salt Agar (MSA), which is differential for mannitol fermentation

EMB Plate

E. coli is seen on the left and E. aerogenes on the right

Bacterial colony morphology

Bacteria grow on solid media as colonies
colony is defined as a visible mass of microorganisms all originating from a single mother cell, when inoculated into appropriate medium containing 2% agar and incubated 18-24 hours in a favorable atmosphere therefore a colony constitutes a clone of bacteria all genetically alike Ideally, the colony is the progeny of one, or at most, a few bacteria A colony will usually contain millions of bacterial cells Colony morphology can sometimes be useful in bacterial identification Colonies are described as to such properties as size, shape, texture, elevation, pigmentation, effect on growth medium

To identify the following colonial characteristics/culture characteristics: WHOLE SHAPE OF COLONY EDGE/MARGIN OF COLONY

ELEVATION OF COLONY (turn the place on end to determine height)

CHROMOGENESIS (pigmentation)

- Some bacterial species form an array of pigments: white, red, purple, etc. Some pigments are contained within the cell (i.e., probably not water soluble) Some pigments readily diffuse throughout the medium (i.e, water soluble) Some pigments fluoresce in UV light

OPACITY OF COLONY:

CONSISTENCY:

transparent (clear), opaque, translucent (almost clear, but distorted visionlike looking through frosted glass iridescent (changing colors in reflected light) butyrous (buttery), viscid (sticks to loop, hard to get off) brittle/friable (dry, breaks apart) Is it easy or difficult to emulsify? Does it form a uniform suspension, a granular suspension, or does not emulsify at all?

EMULSIFIABILITY OF COLONY:

Microscopic morphology
Provide presumptive identification of an organism Bacterial Morphology
Bacterial cell is a fundamental unit of any living organism All its functions are genetically controlled and performed by that particular cell structure whether it be physiologic or biochemical Bacteria and other microorganism are usually transparent, which makes the study of the morphologic detail difficult when they are examined in the natural state Routinely used to determine: shape arrangement staining reaction

I. Bacterial Shape and Arrangement

Bacterial Shape determined by the configuration of the cell wall detected by brightfield microscopy of stained smear Bacterial Arrangement is the result of the number of plane division the organism may undergo and how the cell remain attached afterwards divides only across their short axis 3 conventional forms :

Spherical (cocci)

Rod (bacilli)

Spirals

Spherical (Cocci)

Shape: round like a ball, perfect sphere or globe Variations : 1. Ovoid shape - both sides rounded ends are pointed Ex. Streptococcus 2. Lancet-shape - one end is pointed, other end is flat Ex. Pneumococcus 3. Coffee-bean shape - flat on one side, opposite side convex or appear as letter D form Ex. Neisseria

Arrangements:
1. Singly occurs as a single spherical cell

2. Chain streptococci - common among ovoid-form resulting from one plane division with daughter cells remained attached to one another to form a chain Ex. Streptococcus pyogenes
3. Pairs diplococci - common with lancet-shaped and coffee-bean shaped spherical resulting from one plane division with daughter cell separating Ex. Streptococcus pneumoniae Neisseria gonorrheae

4. Clusters staphylococci
- common with spherical resulting from many plane division with daughter cell in grape-like agglomeration Ex. Staphylococcus aureus

5. Tetrads (Packets of 4) - result from 2 plane division with daughter cell

separating from one another to form group of 4 cells Ex. Micrococcus tetragenous

6. Sarcinae (Packets of 8) - results from many plane division producing cubical

packets of 8 cells Ex. Sarcina lutea

Shape cell appears longer than wide or cylindrical form both sides parallel and ends are convex varies in actual form depending on the species divides only across their short axis Variations : 1. Clubbed/drumstick shaped swollen on one end Ex. Clostridium diphtheriae/C. tetani 2. Corset-shape both sides swollen, ends flat or concave Ex. Bacillus anthracis 3. Fusiform both sides parallel, ends pointed

Rods (Bacilli)

Arrangements: 1. Singly occurs as a single rod 2. Chain result from one plane division with daughter

cell remain attached to one another Ex. Bacillus anthracis 3. Palisade arrangement like fence due to slipping movement of daughter cells (side-by-side) Common among clubbed shaped rods Ex. Mycobacterium tuberculosis 4. Chinese-letter common with clubbed-shaped rods resulting from a snapping post division movement of the daughter cells (V shape) Ex. Corynebacterium diptheriae 5. Packets of cigarette arrangement like bundles Ex. Mycobacterium leprae 6. Serpentine commonly seen with virulent strain of Mycobacterium tuberculosis

Intermediate forms

Coccobacilli
- when a rod is short & wide/plump - these form is intermediate between a spherical and rod Ex. Haemophilus, Brucella

Vibrio
- a gently curve bacteria (comma-shaped) - it is an intermediate between a rod and a spiral Ex. Vibrio cholerae

Spirals
1.

bacteria with more than one somatic curve may be regarded as bacillary forms trusted in the form of a helix no characteristic cell arrangement most occurs singly different specie vary in size, length, rigidity and amplitude of their coils 2 types : Flexible spirals that can contract and relax & move by creeping movement Ex. Spirochetes 2. Rigid spirals that cannot contract and relax & move by rotation or corkscrew-like motion
Ex. Spirillum

SPIRILLUM
- whose long axis remains rigid when in motion Ex. Campylobacter jejuni

SPIROCHETE
whose long axis bends when in motion
Genus Treponema char. tightly coil w/ cork screw appearance Ex. Trepanema pallidum Genus Leptospira less tightly coiled w/ sharp hook-like bends Ex. Leptospira interrogans Genus Borrelia much less tightly coiled w/c has the appearance of extremely long undulating bacillary pores Ex. Borrelia recurrentis

II. Bacterial size

all linear measurements in microbiology are expressed in metric units the basic unit of the metric system is the meter m centimeter cm (1/100th of a m) - the largest unit of length used for measuring microorganism micrometer m - visible only with high powered microscope - unit of measurement most frequently used in microbiology 1m = 1/1000 of a mm Cocci = 0.4-2m Bacilli = 0.2-4m in width by o.5-20m in length Spirals = 1-4m in length nanometer nm - commonly used to measure virus Angstrom smallest unit of measurement

III. Bacterial

Staining Reaction

Staining procedure that applies colored chemicals called dyes to specimen in order to facilitate identification Stains - salts composed of a positive and negative ion, one of which is colored (chromophore color bearing ion), which imparts a color to cell or cell parts by becoming affixed to them through a chemical reaction Basic (cationic) Dyes - chromophore is the positive ion dye Acid (anionic) Dyes - chromophore is the negative ion dye
Bacteria are slightly negative, so are attracted to the positive chromophore of the BASIC DYE

 Preparing

smears for staining

1.

Smear preparation - depends on the physical state; if in liquid state spread the smear out - Bacteria on slide 2. Air Dry - preserve the morphology of the bacteria - allow the smear to adhere to the slide 3. Bacteria are HEAT FIXED to the slide Heat Fixation - simultaneously kills the specimen and secures it to the slide - preserve various cellular component in a natural state with minimal distortion 4. Stain is applied Staining coloring the microorganisms with a dye

Types of Staining: 1. Simple Staining

- employs one dye - most common: methylene blue, crystal violet, carbol fuchsin,safranin - sufficient to determine size, shape & arrangement - most cells will stain the same color with the dye used

Positive Staining Appearance of organisms

Colored by dye

Negative staining
Clear and colorless

Background

Not stained

Stained

2. Differential Staining
- employs the use of more than one dye added in several steps and stained structures are differentiated by color as well as shape - it is based on the relative affinity of different bacterial cells for the stains used - enables microbiologist to differentiate one group from another a) Gram staining - differentiate gram (+) from gram (-) bacteria b) Acidfast staining - differentiate acidfast from non-acidfast bacteria

Gram-staining

Hans Christian Gram (1884), a Danish doctor, accidentally stumbled on a method which still forms the basis for the identification of bacteria; which divided almost all bacteria into two large groups The reagents needed: Crystal Violet (Primary Stain) Iodine Solution (Mordant)

Mordant - intensifies the stain or coats a structure to make it thicker and easier to see after it is stained - Increase the affinity of a stain to the specimen

Decolorizer (ethanol is a good choice, mixture of acetone & alcohol) Safranin (Counterstain)

Counterstain gives contrasting color to the primary stain

Gram Staining STEP 1: Make a smear. Mounted and heat fixed. STEP 2: Flood the entire slide with crystal violet (primary stain) for 1min. Then rinse with the water. STEP 3: flood the slide with the iodine solution (mordant) for 1min. Then rinse with water for 5 seconds. The bacteria become deeply stained and appear deep purple in color due to crystal violet-iodine-complex formation

Step 4: addition of the decolorizer, 95% ethanol. Rinse with water. Gram (+) cells : purple dye is retained Gram (-): purple dye is readily removed and appears colorless STEP 5: Flood the slide with the counterstain, safranin Again, rinse with water. Gram (+) cells will incorporate little or no counterstain and will remain purple in appearance Gram (-) bacteria take on a pink/red color

Divides bacteria into 2 groups Gram (+) : violet Gram (-) : red

Dictome of Gram Staining All COCCI are Gram Positive except Neisseria group, Moraxella (Branhamella) catarrhalis and Veilonella All BACILLI are Gram Negative except the acid fast organisms (Mycobacterium, Nocardia) , Sporeformers (Bacillus, Clostridum) and Corynebacterium species Spirals are difficult to stain but when stained, they are Gram Negative

Gram nagative bacilli

Gram positive cocci

Acid Fast Staining

Acid-fast stain is a useful differential staining procedure that specifically stains all members of the genera mycobacteria The walls of certain bacteria contain long chain fatty acids (mycolic acid) lending the property of resistance to decolorization of basic dyes by acid alcohol; thus called acid fast The high lipid and wax content of the mycobacterial cell walls is thought to be the reason for such impermeability 2 methods Ziehl-Neelsen method

The procedure utilizes heat and phenol (carbolic acid) to help the penetration of the dye, carbol fuchsin, to the inside of mycobacterial cells, which are impermeable to basic dyes in routine stains like in Gram staining Instead of heat, this technique uses increasing the concentration of phenol or the inclusion of a detergent in the stain

Cold Kinyoun technique

 Divides
Acid

bacteria into 2 groups

- Fast organism: red Non Acid Fast organism: blue

The

reagents needed 1. Primary stain: Carbol fuchsin 2. Decolorizer: Acid Alcohol 3. Counterstain: Methylene Blue

Acid - Fast Staining (Ziehl-Neelsen method)

STEP 1: Make a smear. Mounted and heat fixed STEP 2: Flood the entire slide with Carbol Fuchsin. STEP 3: Using a Bunsen burner, heat the slides slowly until they are steaming. Acid fast organisms have a very hydrophobic surface which resist entry of dyes. Heat is used to enhance penetration and retention of dye Maintain steaming for 5 minutes by using low or intermittent heat (i.e. by occasionally passing the flame from the Bunsen burner over the slides) Then rinse the slide with water. STEP 4: Flood the slide with 3% acid-alcohol and allow to decolorize for 5 minutes. Throughout the 5 minutes, continue to flood the slides with 3% acid-alcohol until the slides are clear of stain visible to the naked eye. Rinse the slide thoroughly with water and then drain any excess from the slides. STEP 5: Flood with the counterstain, Methylene Blue Keep the counterstain on the slides for 1 minute. Rinse with water.

3. Special Staining
- used to color and isolate specific structure of a microorganism like capsule, flagella, inclusion granule, endospore and etc.

Positive Staining Capsule

Negative staining

Flagella

Endospore

Biochemical Test

various species of organism exhibits characteristic pattern of substrate utilization, metabolic product formation and sugar fermentation Enzyme based test based on its reaction with a substrate

Catalase, oxidase, indole, urease

Reactions in glucose fermentation broth Reactions in lactose fermenation broth Starch hydrolysis of test strains Nitrate Broth reactions 60% of common pathogens can be identified by metabolic test

Biochemical tests for identification of enteric organisms

Basis: metabolic action of microorganisms on the culture media Used for the identification of enteric organisms/ gram negative bacilli

One of the earliest sets of test used for the identification of enteric bacilli includes such organisms as Klebsiella, Enterobacter, Citrobacter and Escherichia coli

This acronym stands for I - Indole M- Methyl red V - Voges Proskauer ( i ) is inserted for euphony C - Citrate

Indole Test

Indole, a benzyl pyrrole, is one of the metabolic degradation product of amino acid tryptophan Indole positive bacteria produce tryptophanase, an enzyme that is capable of hydrolyzing and diaminating tryptophan, thus producing: - indole - pyruvic acid - ammonia Materials: 2% Peptone broth tube Test organisms Ether indicator: Erlich/Kovac's reagent (para-dimethyl-aminobenzaldehyde)

Indole Medium

Tryptone broth, contains extra tryptophan Trp (tryptophan) can be utilized as a sole carbon and energy source by some bacteria that produce an enzyme tryptophanase Tryptophan is significant because can be directly incorporated into proteins, or can be broken down by organisms with tryptophanase
(tryptophanase)

Tryptophan

Indole + pyruvic acid + NH3

(C-source) (N-source)

The presence of bi-product Indole can be tested with Korvacs reagent Korvacs reagent reacts with Indole and turns solution red

 Procedure:

Inoculate 1 loopful of the test organism into the tube of peptone broth.
Incubate at 370C for 24-48 hours. add 1 ml. of ether. Shake well and allow to stand for a few minutes until the ether rises to the surface. Gently add about, 1cc. of Kovacs or Erlichs reagent down the side of the tube so that it forms a ring between the medium and the ether layer.

Positive result Bright red or purple ring If indole has been produced by the organism it will, being soluble in ether, it will be concentrated in the ether layer and upon the addition of Erlichs reagent, a positive result is the production of a purple ring at the junction of the medium and the ether layer Negative result Yellow color - no red or purple ring

Methyl red and Voges-Proskauer (MR/VP)

Both tests are performed in the same medium (in same tube!!) This medium is used to detect both mixed acid fermentation and 2,3 butanediol fermenters Strains of E. coli are mixed acid fermenters; they degrade carbohydrates into acidic end products such as: lactic acid, acetic acid, succinic acid, and formic acid

The Methyl-Red tests for acidic products resulting from fermentation These acidic products will drop the pH of the medium to pH 4.5 or below Methyl red pH indicator will turn red if mixed acid fermentation has occurred

MR/VP (continued)

The Enterobacter-Klebsiella groups produce ethanol and 2,3 butanediol

The Voges-Proskauer test determines the presence of acetyl-methyl-carbinol (acetoin), which is a precursor to 2,3 butanediol The presence of 2,3 butanediol cannot directly be determined Peptone Glucose Buffer

MRVP broth has several components:

MR/VP is a fermentation test for glucose

Usually, the results are opposite for MR and VP EX:

Fermentation (acidic end products) (alcoholic end products) MR+ (VP-) VP+ (MR-) Glucose Fermentation

No fermentation

(no acid or alcohol products)

(MR- & VP-)

MR (+) bacteria can produce lactose, succinate, formate, and acetate VP (+) bacteria can produce acetoin, acetyl-methyl carbinol, ETOH

Methyle red test

All enterics oxidize glucose for energy; however the end products vary depending on bacterial enzymes Both the MR and VP tests are used to determine what end products result when the test organism degrades glucose MR test is a quantitative test for acid production, requiring positive organism to produce strong acids (lactic, acetic, formic) from glucose via the mixed acid fermentation pathway End result is based on the final pH reached only those organism that can maintain low ph of about ph 4-4.5 can be called methyl red positive organisms that are MR (+) are always VP (-)

 Materials:

MR-VP broth medium (contains 10% glucose) Test organisms Methyl red ph indicator

 Procedure:

Inoculate 1 loopful of the test organism into a tube MR-VP medium. Incubate for 24-48 hours at 370C. Next laboratory period, add 5 to 10 drops of methyl red reagent. Mix thoroughly and observe the results.

Positive result cherry red/bright red color ph 4-4.5 Ex. Salmonella, Escherichia, Citrobacter, Proteus, Morganella and Providencia Negative result Yellow color At neutral pH the growth of the bacteria is not inhibited The bacteria thus begin to attack the peptone in the broth, causing the pH to rise above 4.5 At this pH, methyl red indicator produce a yellow color Ex. Enterobacter and Klebsiella

Voges Proskauer test

is a test for the detection of acetyl-methyl carbinol (acetoin) which is also a degradation product of glucose Materials: MR-VP medium (contains 10% glucose) Test organism Potassium Hydroxide Alpha-napthanol reagent When these reagents are added to a broth in which acetyl methyl carbinol is present, they turn a burgundy color/crimson red color (a positive VP test) organisms that are VP (+) are always MR (-)

Procedure

Inoculate MR-VP medium with 1 loopful of the test organism Incubate for 48 hours at 370C. Add 0.6 ml. 5% alpha-napthol reagent. Mix and shake the mixture lightly. Add 0.2 ml (5drops). of 40% potassium hydroxide reagent
(KOH).

Mix and shake the mixture lightly. Shake the tube gently to expose the medium to atmospheric oxygen and allow the tube to remain undisturbed for 10 to 15 minutes.

 Positive
Crimson

result

Red color Presence of Acety methyl carbinol Ex. Enterobacter and Klebsiella
Negative
Remains

result

Yellow to Amber; no change in color Ex. E. coli

Citrate Test

Simmons Citrate agar is used to determine an organisms ability to use citrate as a sole carbon source Simons citrate agar is a defined medium in which sodium citrate is the sole carbon source, and ammonium is the sole nitrogen source Bromothymol blue (BTB) is included as a pH indicator The medium is initially at pH 6.9, at which BTB is green; at a pH greater than 7.6 BTB turns a deep blue

Citrate Test (continued)

The pH change is induced by CO2, which is given off as a by-product of citrate utilization. When it reacts with Na and H2O in the agar it raises the pH above 7.6 The organism must contain the enzyme citrase to degrade citrate EX:
(citrase)

Citrate

CO2 + Na HCO + H2O

(blue color change)

Procedure:
Inoculate the test organism on the medium by stab streaking. Incubate at 370C for 24 - 48 hours. Observe.

Positive result Deep blue/ Prussian blue color indicating that the test organism has been able to utilize citrate for energy source Ex. Enterobacter, Klebsiella, Salmonella, Citrobacter and Providencia

Negative result Retains its original color (Green) Ex. Escherichia, Shigella and Morganella

Motility Test
The two tubes on the left contain a nonmotile bacterial species. Notice the clearly visible line of growth (streak line). The two tubes on the right contain a motile bacterial species. Notice the cloudy media and the less distinct line of growth.

Molecular differentiation

Genomics

Gene characterization Sequencing PCR Hybridization % guanine + cytosine

Serological procedure

Antigen and antibody determination Serological Tests Use group specific antiserum isolated from the plasma of animals that have been sensitized to the organism

The antiserum contains antibody proteins that react with antigens on the unknown organism.

Procedures: agglutination, precipitation test, hemagglutination inhibition, complement fixation, ELISA, Western blot assay Advantages:

Highly specific Does not usually require the organism to be isolated into pure culture Can be used to identify organisms that cant be grown on medium

Antibiotic sensitivity

antibiotic sensitivity is a term used to describe the susceptibility of bacteria to antibiotics Antibiotic susceptibility testing (AST) is usually carried out to determine which antibiotic will be most successful in treating a bacterial infection in vivo Methods of testing: Broth dilution

The lower the dilution, the greater the antibiotic content

Agar dilution Disk diffusion

the Kirby-Bauer test for antibiotic susceptibility, called the disc diffusion test, is a standard that has been used for years

 The bacterium is swabbed on the agar

and the antibiotic discs are placed on top

The antibiotic diffuses from the disc into the agar in decreasing amounts the further it is away from the disc Bacteria are not able to grow around antibiotics to which they are sensitive If the organism is killed or inhibited by the concentration of the antibiotic, there will be NO growth in the immediate area around the disc: called the zone of inhibition The zone sizes are looked up on a standardized chart to give a result of sensititive, resistant, or intermediate

Many charts have a corresponding column that also gives the

Indicator organism

coliform
Family Enterobacteriaceae. Lactose fermenting Intestinal tract of animals Free living in the environment Opportunistic pathogens

Fecal coliforms
A subset of coliforms Indicator organism for fecal pathogens

Escherichia coli

Predominant fecal coliform Indicates potential presence of enteric pathogens

Methods
Multiple-Tube Fermentation Membrane Filter Technique Chromogenic Substrate Test

Membrane Filter Technique

The plate in the upper left shows typical dark blue fecal coliform colonies on a membrane filter after incubation on M-FC agar. The plate in the lower center (purple) is M-FC agar before use, and the plate in the upper right (blue) is a control plate streaked with an E.coli culture.

Rapid, quantitative analyses of coliforms and E.coli, based on COLILERT cultivation (IDEXX, U.S.A.)